Suppression of angiotensin converting enzyme 2, a host receptor for SARS-CoV-2 infection, using 5-aminolevulinic acid in vitro

Angiotensin converting enzyme 2 (ACE2), an entry receptor found on the surface of host cells, is believed to be detrimental to the infectious capability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Scientists have been working on finding a cure since its outbreak with limited success. In this study, we evaluated the potential of 5-aminolevulinic acid hydrochloride (ALA) in suppressing ACE2 expression of host cells. ACE2 expression and the production of intracellular porphyrins following ALA administration were carried out. We observed the reduction of ACE2 expression and intracellular porphyrins following ALA administration. ALA suppressed the ACE2 expression in host cells which might prevent binding of SARS-CoV-2 to host cells. Co-administration of ALA and sodium ferrous citrate (SFC) resulted in a further decrease in ACE2 expression and increase in intracellular heme level. This suggests that the suppression of ACE2 expression by ALA might occur through heme production. We found that the inhibition of heme oxygenase-1 (HO-1), which is involved in heme degradation, also resulted in decrease in ACE2 expression, suggesting a potential role of HO-1 in suppressing ACE2 as well. In conclusion, we speculate that ALA, together with SFC administration, might serve as a potential therapeutic approach in reducing SARS-CoV-2 infectivity through suppression of ACE2 expression.


Introduction
Incidents of patients with pneumonia of unidentified causes were reported in large numbers in late 2019 [1]. The pathogen responsible was later identified in local hospitals as severe acute respiratory syndrome-corona virus-2 (SARS-CoV-2) [2]. Patients infected by SARS-CoV-2 were named as coronavirus disease 2019 (COVID-19) patients by WHO [3]. As of August 2022, 581,686,197 confirmed cases of COVID-19, including 6,410,961 deaths have been reported globally to WHO [3]. Despite scientists' continuous efforts around the world, there is still no effective cure for COVID-19 besides employing clinical managing strategies and providing supportive care for severely ill patients [4,5]. Angiotensin-converting enzyme 2 (ACE2), a receptor found on the surface of human cells, plays a pivotal role in renin-angiotensin-aldosterone system, regulating blood pressure and electrolyte homeostasis [6]. However, the high affinity of viral spike protein of SARS-CoV-2 towards ACE2 has allow viral entry into human cells through S-protein priming by the host cell protease, TMPRSS2 [6]. It is also reported that the binding affinity of S-protein and ACE2 correlate with the replication rate of the virus and severity of the disease [7]. The findings also correlate with the reports where high ACE2 expression leads to higher viral load in nasal swabs and higher disease severity of COVID-19 patients [8,9]. Interestingly, the study by Bunyavanich et. al., (2020), showed that children under age 18 have significantly lower ACE2 expression compared to adults, suggesting low SARS-CoV-2 infectivity among children [10]. Therefore, it is believed that the suppression of ACE2 may be a novel method to reduce infectious capability of SARS-CoV-19.
5-Aminolevulinic acid (ALA) is an essential amino acid, with little toxicity, naturally found in the human body [11]. The conjugation of eight molecules of 5-Aminolevulinic acid lead to the formation of protoporphyrin IX (PpIX), which may be converted in heme prior to the insertion of ferrous ion (Fig 1) [12]. Since administration of ALA lead to heme formation, which is essential in maintaining the optimum function of protein complexes such as cytochromes, ALA might be used to improve metabolic processes in various diseases such as diabetes [13]. In addition, ALA has been used in photodynamic diagnosis and photodynamic therapy of various cancers at a very high dose such as 20 mg/kg body weight which might cause some side effects [14][15][16][17][18]. A preliminary study by Sakurai et al. (2021) [19], suggested the reduced infectivity of SARS-CoV-2 following ALA administration, although the molecular mechanisms leading to this phenomenon were not yet evaluated [20]. We hypothesized that ALA might inhibit infectivity of SARS-CoV-2 by suppressing the expression of ACE2 which is a host cell receptor for S-protein of SARS-CoV-2, possibly due to increase in heme production following ALA administration. We studied the mechanism of 5-Aminolevulinic acid in inhibiting SARS-CoV-2 infectivity.

Cells and cell culture
The human liver cancer cell line, HepG2, was provided by SBI Pharmaceuticals Co. Ltd. (Tokyo, Japan). VeroE6, a cultured cell line derived from kidney epithelial cells isolated from African green monkeys, was obtained from JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition, Japan. The cells were cultured in DMEM (Low) culture medium containing 10% foetal bovine serum (FBS) and 10% ABAM, at 37˚C in a 5% CO 2 incubator. Experiments were performed at a cell density of 50%-80% confluence.

HPLC analysis of PpIX and heme
Quantification of PpIX and heme by HPLC were carried out by preparing cell lysate through administration of 0.1 M NaOH to PBS-washed samples. A protein denaturant equivalent to 3 times of the cell lysate was added to extract PpIX and heme in the cells. The protein denaturant was a mixture of mobile phase A (1 M ammonium acetate solution containing 12.5% acetonitrile adjusted to pH = 5.2) and mobile phase B (50 mM ammonium acetate solution containing 80% acetonitrile) solution at a ratio of 1:9 (v/v). Centrifugation was performed twice at 10,000×g for 10 min at 4˚C to remove denatured proteins, and the supernatant was collected. Protein concentrations were quantified by the Bradford method using Quickstart™ Bradford 1×Dye Reagent (Bio-rad Laboratories Inc., California, USA). For HPLC analysis of PpIX and heme, a Type Prominence system (Shimadzu, Kyoto, Japan) were used. Mobile phase A and mobile phase B were used for elution of porphyrins. The elution program consisted of 10% mobile phase A and 90% mobile phase B at a flow rate of 2 mL/min for 7 minutes. The eluate was measured by absorbance at 404 nm using a spectroscopic detector. 100 μL of sample was injected. Hemin used as a heme standard was purchased from Nacalai Tesque (Kyoto, Japan). Protoporphyrin IX dihydrochloride used as a standard substance for PpIX was purchased from Frontier Scientific, Inc. (UT, USA).

Statistical analysis
Microsoft Excel 2010 was used for data analysis in this study. A one-way ANOVA (Tukey's Test) was performed for each data set to identify differences in mean values between treated and non-treated samples; at two levels of significance, p < 0.05 and p < 0.01.

Effect of ALA on ACE2 expression in HepG2 and VeroE6 cells
In order to study the effect of ALA administration on infectious capability of SARS-CoV-2, we first studied the changes in expression of ACE2, a surface receptor which binds to spike protein of SARS-CoV-2. HepG2 and VeroE6 cells were incubated at 2.15 × 10 4 cells / cm 2 at 37˚C in 5% CO 2 -atmosphere for 48 h before evaluating the change in protein expression level of ACE2 using Western blot. Fig

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Suppression of ACE2, a SARS-CoV-2 infection related protein, using 5-aminolevulinic acid in vitro of ACE2 following ALA administration in VeroE6 cells, a cultured cell line commonly used in SARS-CoV-2 study. These results suggested ALA administration led to decrease in availability of ACE2, a surface receptor for SARS-CoV-2, on host cells.

Changes in intracellular PpIX and heme levels following ALA administration
After its entry into the cell, ALA is converted into PpIX and heme through a series of enzymemediated reactions in the cell [22,23]. The uptake of ALA into the cell can be determined by measuring the concentration of PpIX and heme produced using high performance liquid chromatography (HPLC) as described in the Materials & Methods. We observed significant increases in the concentration of intracellular PpIX in both HepG2 and VeroE6 cell lines (Fig 3). This suggest that ALA was uptaken into the cell and converted into PpIX. However, only a slight increase (statistically insignificant; p > 0.05) in the heme concentration was observed. We hypothesized that this might be due to the lack of iron source which limited the conversion of PpIX into heme.

Changes in ACE2 expression following ALA and SFC co-administration
Sodium ferrous citrate (SFC) is commonly used an iron source in various studies [24,25]. In order to induce more conversion of PpIX into heme, we evaluated the effect of co-administration of ALA and SFC on ACE2 expression. Coincided with results shown in Fig 2, ALA administration resulted in decrease in ACE2 expression (Fig 4). The co-administration of ALA and SFC further reduced the expression levels of ACE2 in both cell lines, particularly in VeroE6 cell line (Fig 4). These findings suggest that SFC may enhance inhibitory effect of ALA on ACE2 expression.

Changes of porphyrin-heme production and ACE2 suppression following co-administration of ALA and SFC
Findings from Fig 4B showed changes of ACE2 expression following the administration of ALA and SFC. In this section, we evaluated the concentration of intracellular PpIX and heme in VeroE6 cells following administration of ALA in the absence and presence of SFC (Fig 5). PpIX level in VeroE6 cells remained undetectable following administration with SFC only. No increase in PpIX production compared to the control was observed (Fig 5A). The co-administration of ALA and SFC resulted in a decrease in PpIX production compared to ALA-treated samples. Fig 5B showed the concentration of heme in VeroE6 cells increased following ALA administration. A slight decrease in heme level was observed following SFC-only treatment. Interestingly, the concentration of intracellular heme increased following co-administration of ALA and SFC. This finding suggests the administration of iron source induce PpIX conversion

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Suppression of ACE2, a SARS-CoV-2 infection related protein, using 5-aminolevulinic acid in vitro into heme, leading to increase in ALA-induced heme production and reduction of ACE2 expression.

Changes of ACE2 expression following ALA administration and HO-1 inhibition
In this section, we attempted to increase heme production by inhibiting heme oxygenase 1 (HO-1) to evaluate its effect on ACE2 expression. HO-1 is important in catalyzing the degradation of heme into biliverdin and bilirubin [26]. Zinc protoporphyrin IX (ZnPpIX) was used as an inhibitor for suppressing HO-1 activity, leading to high production of heme [27]. Fig 6 showed the changes of protein expression level of ACE2 following ALA and ZnPpIX administration. Coincided with the results shown in Fig 2A, the expression of ACE2 decreased following ALA administration. Moreover, the treatment of HepG2 with ZnPpIX showed a further decrease in ACE2 expression. Our findings suggest that porphyrin and/or heme play a role in inhibiting ACE2 expression, and that the administration of ALA inhibit ACE2 expression through porphyrin and/or heme production (Fig 7).

Discussion
Aside from clinical managing strategies and supportive care for COVID-19 patients, there is currently no effective cure for treating the disease [4,5]. The emergence of new SARS-CoV-2 variants which possess higher infectivity and stronger resistance towards currently available vaccines only further worsen the situation [28]. ACE2, a receptor found on the surface of human cells, allows the virus to enter the host cell after binding to viral spike protein [5]. While ACE2 is known to be highly expressed in nasal epithelial cells, it is also reported that the

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Suppression of ACE2, a SARS-CoV-2 infection related protein, using 5-aminolevulinic acid in vitro expression of ACE2 increases as people aged [7,10]. This is particularly dangerous since most elderly people have weaker immune system compared to younger populations [10]. Therefore, it is imperative that scientists need to develop novel drugs (e.g., ACE2-targeting drugs) to fight against SARS-CoV-2.
5-Aminolevulinic acid, a natural amino acid which is highly useful in treating various kinds of diseases through improvement of the body's metabolic activity, has showed the potential to exert its inhibitory effect on the infectivity of SARS-CoV-2 in vitro although the exact mechanism has not yet been evaluated [19,20]. We hypothesized that ALA possesses its inhibitory effect on ACE2 expression in host cells. Our results shown in Fig 2 demonstrated that the expression of ACE2 was significantly reduced in both cell lines of HepG2 and VeroE6 following ALA administration. The uptake of ALA was shown by the increased of PpIX level (Fig 3) although the level of heme only showed a slight increase (statistically insignificant; p > 0.05) in

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HepG2 cells. We hypothesized that the increase in heme level might be limited due to the lack of ferrous iron because the insertion of ferrous ion is essential in converting PpIX into heme. Our hypothesis was proven by the observation that the administration of SFC, which acts as an iron source, led to a further decrease in ACE2 expression compared to ALA-only treated samples, particularly in VeroE6 cell line (Fig 4). We believed the addition of SFC affect iron metabolism in the cells which somehow lead to reduction in ACE2 expression, although the exact mechanism remain unclear. This further decrease in ACE2 expression suggest that the effectiveness of ALA in inhibiting infectivity of SARS-CoV-2 might be enhanced by heme production.
It is known that RNA G-quadruplex within transmembrane serine protease 2 and ACE2 of the host cell both play important roles in preventing SARS-CoV-2 entry [29]. In year 2018, Shioda et al. showed PpIX is likely interfere with interaction of G-quadruplex structures [30]. In addition, the Japanese COVID-19 patients who were orally administered with ALA and SFC capsules showed a shorter recovery time than patients which underwent standard care for SARS-CoV-2 infection [31]. We hypothesized the addition of ALA and SFC would in turn generate PpIX production that would interfere with G-quadruplex structures and ACE2,

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Suppression of ACE2, a SARS-CoV-2 infection related protein, using 5-aminolevulinic acid in vitro leading to reduced infectivity by SARS-CoV-2. In Fig 5, the roles of PpIX and heme in the expression of ACE2 were determined by evaluating the concentration of intracellular PpIX and heme following ALA and SFC co-administration. The decrease in PpIX level following coadministration of ALA and SFC suggested that some of PpIX were converted into heme molecules in an iron source-abundant environment (Fig 5A). Fig 5B showed that the heme level was elevated following the co-administration of ALA and SFC. In accordance with the finding shown in Fig 5A, the increase in heme level is assumed to be a result of increasing conversion of PpIX to heme following the insertion of ferrous ion. Kim et. al. (2021) showed that hemin administration suppressed the replication of SARS--CoV-2 [32]. Hemin is an exogenous source of heme, which is commonly used to treat porphyria-related diseases [33]. Our findings from Figs 4B and 5B suggest the concentration of intracellular heme play an important role in regulating the expression of ACE2. The administration of ALA, which induced an increase in heme level, has been observed to lead to a decrease in the expression of ACE2. In order to prove that ALA administration suppressed SARS-CoV-2 infectivity through elevated heme production, we studied the effect of HO-1 inhibition on ACE2 expression level. HO-1, a heme-degrading enzyme, catalyses the degradation of heme into biliverdin and bilirubin [25]. Inhibition of HO-1 might result in an increase in heme level. As shown in Fig 6, the increase in heme level correlated with the enhancement of the suppressive capability of ALA on the expression of ACE2, suggesting the strong relationship between heme and ACE2 expression. This shows the importance of heme in suppressing ACE2 expression, which prevents binding of viral spike proteins to host cells, and ultimately leads to lowered infectivity of SARS-CoV-2. As an agent which stimulates the porphyrin-heme synthesis pathway and increase heme production, ALA might be a potential drug for preventing infiltration of host cell by SARS-CoV-2 through inhibition of ACE2 expression. In addition, it is important to note that the administration of HO-1 inhibitor, ZnPpIX, showed significant decrease in ACE2 expression (Fig 6), suggesting the expression of HO-1 may also contribute to the change in expression of ACE2 and the infectivity of SARS-CoV-2, although the exact mechanism behind this remain unknown.
In this study, we hypothesized the usage of ALA as a potential anti-viral drug targeting ACE2 for SARS-CoV-2 patients. The nature of ALA administration in human such as the safety is reported based on its application in photodynamic diagnosis for fluorescent guided surgery for tumour resection. Therefore, developing ALA as an anti-viral agent should be realistic plan. On the other hand, ACE2 is known to modulate blood pressure homeostasis in human. Although there were initial concerns regarding the usage of ACE inhibitor as drugs for treating COVID-19 patients, there is currently no scientific evidence which suggests it is potentially harmful to administered patients.
In conclusion, ALA administration to the cells induced higher heme production when iron source was available, resulting in inhibition of ACE2 expression, which could lead to prevention of viral spike protein binding to host cells. However, the direct role of PpIX and/or heme in the inhibition of ACE2 binding to viral spike proteins might be important as well although further studies are required. We suggest ALA as a potential anti-viral agent for SARS-CoV-2 which might play an important role in the eradication of the disease in a global scale in the near future.